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• W2006569711 abstract "Ras activates three mitogen-activated protein kinases (MAPKs) including ERK, JNK, and p38. Whereas the essential roles of ERK and JNK in Ras signaling has been established, the contribution of p38 remains unclear. Here we demonstrate that the p38 pathway functions as a negative regulator of Ras proliferative signaling via a feedback mechanism. Oncogenic Ras activated p38 and two p38-activated protein kinases, MAPK-activated protein kinase 2 (MK2) and p38-related/activated protein kinase (PRAK). MK2 and PRAK in turn suppressed Ras-induced gene expression and cell proliferation, whereas two mutant PRAKs, unresponsive to Ras, had little effect. Moreover, the constitutive p38 activator MKK6 also suppressed Ras activity in a p38-dependent manner whereas arsenite, a potent chemical inducer of p38, inhibited proliferation only in a tumor cell line that required Ras activity. MEK was required for Ras stimulation of the p38 pathway. The p38 pathway inhibited Ras activity by blocking activation of JNK, without effect upon ERK, as evidenced by the fact that PRAK-mediated suppression of Ras-induced cell proliferation was reversed by coexpression of JNKK2 or JNK1. These studies thus establish a negative feedback mechanism by which Ras proliferative activity is regulated via signaling integrations of MAPK pathways. Ras activates three mitogen-activated protein kinases (MAPKs) including ERK, JNK, and p38. Whereas the essential roles of ERK and JNK in Ras signaling has been established, the contribution of p38 remains unclear. Here we demonstrate that the p38 pathway functions as a negative regulator of Ras proliferative signaling via a feedback mechanism. Oncogenic Ras activated p38 and two p38-activated protein kinases, MAPK-activated protein kinase 2 (MK2) and p38-related/activated protein kinase (PRAK). MK2 and PRAK in turn suppressed Ras-induced gene expression and cell proliferation, whereas two mutant PRAKs, unresponsive to Ras, had little effect. Moreover, the constitutive p38 activator MKK6 also suppressed Ras activity in a p38-dependent manner whereas arsenite, a potent chemical inducer of p38, inhibited proliferation only in a tumor cell line that required Ras activity. MEK was required for Ras stimulation of the p38 pathway. The p38 pathway inhibited Ras activity by blocking activation of JNK, without effect upon ERK, as evidenced by the fact that PRAK-mediated suppression of Ras-induced cell proliferation was reversed by coexpression of JNKK2 or JNK1. These studies thus establish a negative feedback mechanism by which Ras proliferative activity is regulated via signaling integrations of MAPK pathways. mitogen-activated protein kinase p38-related/activated protein kinase MAPK-activated protein kinase 2 extracellular mitogen-regulated kinase c-Jun NH2-terminal kinase (also called stress-activated protein kinase, SAPK) MAPK kinase JNK or SAPK kinase cytomegalovirus glutathione S-transferase hemagglutinin topoisomerase IIα heat shock protein β-galactosidase myelin basic protein serum response element Ras regulates multiple signaling pathways (1Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (918) Google Scholar). Of these the best described are the mitogen-activated protein kinases (MAPKs),1 including extracellular mitogen-regulated kinase (ERK), Jun amino-terminal kinase (JNK, also called stress-activated protein kinase, SAPK), and p38 (2Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (470) Google Scholar). Through interaction with Raf proteins, Ras activates MEK1 and MEK2, which in turn activate ERK (3Westwick J.K. Cox A.D. Der C.J. Cobb M.H. Hibi M. Karin M. Brenner D.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6030-6034Crossref PubMed Scopus (165) Google Scholar, 4Khosravi-Far R. Solski P.A. Clark G.J. Kinch M.S. Burridge K. Der C.J. Mol. Cell. Biol. 1995; 15: 6443-6453Crossref PubMed Scopus (638) Google Scholar, 5Downward J. Curr. Opin. Gen. Dev. 1998; 8: 49-54Crossref PubMed Scopus (503) Google Scholar). Whereas Raf-independent transduction of Ras signaling was reported (6Gangarosa L.M. Sizemore N. Graves-Deal R. Oldham S.M. Der C.J. Coffey A.J. J. Biol. Chem. 1997; 272: 18926-18931Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 7Klesse L.J. Meyers K.A. Marshall C.J. Parada L.F. Oncogene. 1999; 18: 2055-2068Crossref PubMed Scopus (181) Google Scholar), the Ras/Raf/MEK/ERK pathway provides a common route by which signals from different growth factor receptors converge to activate major transcription factors such as AP1. Ras also activates JNK, which also plays a role in regulating AP1 activity (8Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2239) Google Scholar). The critical role of the JNK pathway in Ras signaling is suggested by the observation that Ras only poorly transforms c-jun null cells (9Johnson R. Spiegelman B. Hanahan D. Wisdom R. Mol. Cell. Biol. 1996; 16: 4504-4511Crossref PubMed Scopus (249) Google Scholar) and blocking of the JNK pathway can inhibit Ras-induced transformation (10Clark G.J. Westwick J.K. Der C.J. J. Biol. Chem. 1997; 272: 1677-1681Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The signaling pathway from Ras to JNK, however, is less clear, although it has been shown that Ras can directly interact with c-Jun proteins, JNK (11Adler V. Pincus M.R. Brandt-Rauf P.W. Ronai Z. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10585-10589Crossref PubMed Scopus (80) Google Scholar), and its upstream activating kinase (MEKK1) (12Russell M. Lange-Carter C.A. Johnson G.L. J. Biol. Chem. 1995; 270: 11757-11760Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Most Ras signaling is believed to be transmitted via these two pathways (8Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2239) Google Scholar). Ras can also stimulate p38, albeit less efficiently (13Whitmarsh A.J. Yang S.H. Su M.S.S. Sharrocks A.D. Davis R.J. Mol. Cell. Biol. 1997; 17: 2360-2371Crossref PubMed Scopus (437) Google Scholar, 14Lin A. Minden A. Martineto H. Claret F. Lange-Carter C. Mercurio F. Johnson G.L. Karin K. Science. 1995; 268: 286-290Crossref PubMed Scopus (706) Google Scholar). The biological consequence of p38 activation in Ras signaling transduction, however, remains unclear. It is likely that even this moderate activation could have an important impact on Ras signaling. p38 is most strongly activated by proinflammatory cytokines and environmental stresses (15Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2390) Google Scholar, 16Lee J.C. Laydon J.T. McDonnel P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3113) Google Scholar). It is activated by the upstream kinases MKK3, MKK6 (14Lin A. Minden A. Martineto H. Claret F. Lange-Carter C. Mercurio F. Johnson G.L. Karin K. Science. 1995; 268: 286-290Crossref PubMed Scopus (706) Google Scholar, 17Han J. Lee J. Jiang Y. Li Z. Feng L. Ulevitch R.J. J. Biol. Chem. 1996; 271: 2886-2891Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 18Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1137) Google Scholar), and MKK4 (also called SEK1 and JNKK1). Several protein kinases have been identified as p38 physiological substrates, including MAPKAP-K2 (MK2) (19Stokoe D. Campbell D.G. Nakielny S. Hidaka H. Leevers S. Marshall C. Cohen P. EMBO J. 1992; 11: 3985-3994Crossref PubMed Scopus (389) Google Scholar), MAPKAP-K3 (20McLaughlin M.M. Kumar S. McDonnel P.C. Van Horn S. Lee J.C. Livi G.P. Young P.R. J. Biol. Chem. 1996; 271: 8488-8492Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), and the recently cloned PRAK (p38-related/activated protein kinase) (21New L. Jiang Y. Zhao M. Liu K. Zhu W. Flood L.J. Kato Y. Parry G.C.N. Han J.H. EMBO J. 1998; 17: 3372-3384Crossref PubMed Scopus (272) Google Scholar). PRAK is a 471 amino acid protein with 20–30% sequence identity to other MAPK-regulated protein kinases (21New L. Jiang Y. Zhao M. Liu K. Zhu W. Flood L.J. Kato Y. Parry G.C.N. Han J.H. EMBO J. 1998; 17: 3372-3384Crossref PubMed Scopus (272) Google Scholar). In addition to phosphorylation and activation of transcriptional factors, some of the p38 pathway-mediated effects may be mediated via these downstream molecules. Whereas the biological consequence of p38 activation may vary under different situations, the following evidence suggests that it may possess anti-mitogenic activity. 1) Although p38 responds to a variety of extracellular stimuli, it is activated most strongly by proinflammatory cytokines (22Miyazawa K. Mori A. Miyata H. Akahane M. Ajisawa Y. Okudaira H. J. Biol. Chem. 1998; 273: 24832-24838Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 23Raingeaud J. Gupta S. Rogers J. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2030) Google Scholar, 24Rincon M. Enslen H. Raingeaud J. Recht M. Zapton T. Su M.S. Penix L.A. Davis R.J. Flavell R.A. EMBO J. 1998; 17: 2817-2829Crossref PubMed Scopus (357) Google Scholar). Consequently, the p38 activation may play a role in controlling proliferation within the immune system. 2) Activation of the p38 pathway was shown to inhibit cyclin D1 expression (25Lavoie J. L'Allemain G. Brunet A. Muller R. Pouyssegur J. J. Biol. Chem. 1996; 271: 20608-20616Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar), which functions downstream of Ras in the direct control of cell proliferation (26Aktas H. Cai H. Cooper G.M. Mol. Cell. Biol. 1997; 17: 3850-3857Crossref PubMed Scopus (367) Google Scholar). 3) The activated p38 pathway is known to phosphorylate and activate Hsp27 (heat shock protein) via MK2in vivo (27Schafer C. Ross S. Bragado M.J. Groblewski G.E. Ernst S.A. Williams J.A. J. Biol. Chem. 1998; 273: 24173-24180Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 28Larsen J.K. Yamboliev I.A. Weber L.A. Gerthoffer W.T. Am. J. Physiol. 1997; 273: L930-L940PubMed Google Scholar), which by itself can inhibit cell proliferation (29Blackburn R.V. Galoforo S.S. Berns C.M. Armour E.P. McEachern D. Corry P.M. Lee Y.J. Int. J. Cancer. 1997; 72: 871-877Crossref PubMed Scopus (26) Google Scholar, 30Arata S. Hamaguchi S. Nose K. J. Cell. Physiol. 1997; 170: 19-26Crossref PubMed Scopus (15) Google Scholar). The signaling mechanisms for this anti-mitogenic effect of p38, however, remain unknown. It is believed that MAPKs regulate target genes by phosphorylation and activation of a group of transcriptional factors such as AP1 and SRE (8Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2239) Google Scholar, 31Hill C.S. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1194) Google Scholar). Recent evidence suggests, however, that signals from three MAPK pathways can be integrated before reaching the transcriptional factors. Activation of the ERK pathway, for example, suppresses p38/JNK-induced apoptosis in PC12 cells (32Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5019) Google Scholar). Inhibition of p38, on the other hand, enhances interleukin 1β-induced expression of the low density lipoprotein receptor, which is inhibited by the MEK inhibitor 98059 (33Kumar A. Middleton A. Chambers T.C. Mehta K.D. J. Biol. Chem. 1998; 273: 15742-15748Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Cross-talk also exists between JNK and p38 pathway. JNK opposes the stimulatory effect of p38 activity on induction of atrial natriuretic factor expression (34Nemoto S. Sheng Z. Lin A. Mol. Cell. Biol. 1998; 18: 3518-3526Crossref PubMed Scopus (209) Google Scholar). Forced expression of constitutively active p38-activating kinase MKK6, however, overcomes apoptosis induced by an active MEKK1, a JNK kinase kinase, in myocardial cells (35Zechner D. Craig R. Hanford D.S. McDonough P.M. Sabbadini R.A. Glembotski C.C. J. Biol. Chem. 1988; 273: 8232-8239Abstract Full Text Full Text PDF Scopus (208) Google Scholar). The possibility thus exists that the activation of p38 by Ras may be able to regulate the output of Ras signaling through interaction with the ERK/JNK pathways. The purpose of this study is to determine how the activation of the p38 pathway may affect Ras proliferative signaling. The HA epitope-tagged wild-type PRAK, the mutant PRAK (PRAK(182D)), and the dominant-negative form of PRAK (PRAK/KM) in pcDNA3 have been previously described (21New L. Jiang Y. Zhao M. Liu K. Zhu W. Flood L.J. Kato Y. Parry G.C.N. Han J.H. EMBO J. 1998; 17: 3372-3384Crossref PubMed Scopus (272) Google Scholar). The HA-tagged wild-type as well as dominant-negative MKK6 (MKK6/2A, with two phosphorylation residues replaced by alanine) have been described previously (17Han J. Lee J. Jiang Y. Li Z. Feng L. Ulevitch R.J. J. Biol. Chem. 1996; 271: 2886-2891Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar). pAC-CMV encoding either activated Ha·Ras (Leu-61) was kindly provided by Dr. Christopher B. Newgard, University of Texas Southwestern Medical Center. pCMV plasmids encoding constitutively active MEK (MEK/2E), dominant-negative MEK-1 (MEK/2A), and dominant-negative SEK1 (SEK/AL), as well as pEBG expressing GST-tagged ERK (p42MAPK) and GST-tagged JNK (p54SAPK) were provided by Dennis Templeton, Case Western Reserve University (36Yan M. Dal T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (658) Google Scholar, 37Yan M. Templeton D.J. J. Biol. Chem. 1994; 269: 19067-19073Abstract Full Text PDF PubMed Google Scholar) and have been previously used in this laboratory (38Chen G. Templeton D. Suttle D.P. Stacey D. Oncogene. 1999; 18: 7149-7160Crossref PubMed Scopus (25) Google Scholar). pcDNA3-MKK6/2E is a constitutively active mutant of MKK6 with Ser-207 and Thr-211 replaced by glutamic acid. This plasmid as well as the FLAG-tagged wild-type MKK6 and pCMV5 vectors containing FLAG-tagged wild-type as well as a kinase dead form of p38α (p38/AGF) were kindly provided by Dr. Roger Davis, University of Massachusetts Medical School (18Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1137) Google Scholar). A mammalian expression plasmid pcDNA3 containing cDNA for Myc epitope-tagged MK2 was supplied by Dr. Matthias Gaestel, Martin-Luther University, Germany (39Engel K. Schultz H. Martin F. Kotlyarov A. Plath K. Hahn M. Heinemann U. Gaestel M. J. Biol. Chem. 1995; 270: 27213-27221Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). HA·JNKK2 and HA·JNK1 in pSRα vector were provided by Anning Lin (40Lu X. Nemoto S. Lin A. J. Biol. Chem. 1997; 272: 24751-247754Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) and Michael Karin (41Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1722Crossref PubMed Scopus (1009) Google Scholar), respectively. Bacterial-expressing plasmid pGEX for GST·Jun was provided by Dennis Templeton, that for GST·ATF2 by Roger Davis, and that for GST·p38/KM has been described before (17Han J. Lee J. Jiang Y. Li Z. Feng L. Ulevitch R.J. J. Biol. Chem. 1996; 271: 2886-2891Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar). These fusion proteins were purified with glutathione-agarose beads (42Smith D.B. Johnson K.S. Gene. 1988; 67: 31-40Crossref PubMed Scopus (5028) Google Scholar). 3AP1 and 5SRE luciferase constructs were kindly provided by Craig Hauser, the Burnham Institute. 3AP1 sequence (43Galang C.K. Der C.J. Hauser C.A. Oncogene. 1994; 9: 2913-2921PubMed Google Scholar) was inserted into a luciferase reporter gene containing a minimal Fos promoter, Δ56FosE-luc (43Galang C.K. Der C.J. Hauser C.A. Oncogene. 1994; 9: 2913-2921PubMed Google Scholar, 44Foos G. Garcia-Ramirez J.J. Galang C.K. Hauser C. J. Biol. Chem. 1998; 273: 18871-18880Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The c-fos SRE sequence was described previously (45Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1197) Google Scholar), and five copies of this was inserted into the same reporter gene (44Foos G. Garcia-Ramirez J.J. Galang C.K. Hauser C. J. Biol. Chem. 1998; 273: 18871-18880Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). DNA topoisomerase IIα (Topo IIα) luciferase promoter in pGL2 vector was provided by P. Suttle (46Wang Q. Zambetti G.P. Suttle D.P. Mol. Cell. Biol. 1997; 17: 389-397Crossref PubMed Google Scholar) and has been used previously in this laboratory (38Chen G. Templeton D. Suttle D.P. Stacey D. Oncogene. 1999; 18: 7149-7160Crossref PubMed Scopus (25) Google Scholar). Dulbecco's modified Eagle's medium and calf serum were obtained from Celox (Hopkins, MN) and Sigma (Sigma), respectively. All other materials for cell culture were supplied by Life Technologies, Inc. DNA was prepared using Endofree kit from QIAGEN and DNA transfection kit (calcium phosphate was purchased from Promega). Luciferase was assayed with a kit from Roche Molecular Biochemicals (Indianapolis, IN). β-galactosidase (β-Gal) was assayed with a kit from Promega. PD 98059 was provided by Alexis cooperation (San Diego, CA). SB203580 was purchased from Calbiochem. Glutathione-agarose beads were from Sigma. Protein G-Sepharose 4B and protein A-Sepharose 4B beads were purchased from Zymed Laboratories Inc. Anti-FLAG M2 affinity gel was bought from Sigma. Anti-HA (clone 12CA5) and anti-c-Myc (clone 9E10) mouse monoclonal antibody was provided by Roche Molecular Biochemicals. Mouse monoclonal antibody against galactosidase was from Promega. Anti-mouse-Cy3 was from Jackson. ATF2-(1–505) for the p38 assay was provided by Santa Cruz Biotechnology, Inc. MBP for the ERK assay was purchased from Sigma, and HSP27 for the PRAK and MK2 assays was obtained from Stress Gen. GST·Jun for the JNK assay was expressed in pGEX (provided by Dennis Templeton) and purified with glutathione-agarose beads (42Smith D.B. Johnson K.S. Gene. 1988; 67: 31-40Crossref PubMed Scopus (5028) Google Scholar). [γ-32P]ATP was from ICN (Costa Mesa, CA). [methyl-3H]Thymidine was from Amersham Pharmacia Biotech. Autoradiography emulsion was from Kodak. The NIH3T3 cell line was obtained from ATCC and maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and antibiotics at 37 °C with 5% CO2. NIH 3T3 line stably transformed by v-ras was provided by Dr. Lowy and has been described previously (47Chen G. Shu J. Stacey D.W. Oncogene. 1997; 15: 1643-1651Crossref PubMed Scopus (55) Google Scholar, 48Stacey D.W. Degudicibus S.R. Smith M.R. Exp. Cell Res. 1987; 171: 232-242Crossref PubMed Scopus (25) Google Scholar). J82 and T24 human bladder carcinoma cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and have been previously characterized in this laboratory for the dependence (T24) and independence (J82) of Ras activity for their proliferation (48Stacey D.W. Degudicibus S.R. Smith M.R. Exp. Cell Res. 1987; 171: 232-242Crossref PubMed Scopus (25) Google Scholar, 49Chen G. Oh S. Monia B.P. Stacey D. J. Biol. Chem. 1996; 271: 28259-28265Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Cells were plated at 1 × 105/ml in 10% fetal calf serum, Dulbecco's modified Eagle's medium 1 day before in 6-well plates for the proliferation assay and in 100-mm dishes for the kinase assay. For microinjection, cells were switched to serum-starved medium (0.5% fetal calf serum) for 48 h before the injection. For transfection, the manufacturer's protocol for calcium phosphate-mediated transfection (Promega) was followed. Total DNA content of 7 μg was used for a 2-ml 6-well plate for the proliferation assay and of 20 μg for a 100-mm dish for the kinase assay and flow cytometry analysis. The next day, DNA-containing medium was removed, and cells were allowed to grow in 0.5% fetal calf serum for 48 h before cells were processed for the different assays. Microinjections were performed into cells within a circular area marked on the back of the coverslip as described previously (50Stacey D.W. Kung H.F. Nature. 1984; 310: 508-511Crossref PubMed Scopus (244) Google Scholar). Plasmid DNA was prepared using endofree kit from QIAGEN and mixed at 1:1:1 ratio (pCMV-β-gal: pAC-Ha·Ras (Leu-61): pcDNA3 or pcDNA3-PRAK or pcDNA3-PRAK/182D or pcDNA3-PRAK/KM. Two DNA concentrations were used, one at 150 μg/ml, another at 450 μg/ml of total DNA (with each at 50 and 150 μg/ml, respectively). Following injection, 5 μCi of [methyl-3H]thymidine was added to each 2-ml plate, and cells were grown in 0.5% fetal calf serum for another 24 h. Cells were then washed twice with phosphate-buffered saline and fixed in 100% methanol at room temperature for 10 min. The rest of the procedures for staining and autoradiography were the same as for the other proliferation assays. For staining and autoradiography, cells were plated on coverslips and transfected or microinjected as above. At the end of the experiments, cells were fixed in methanol and blocked with 3% bovine serum albumin in phosphate-buffered saline for 45 min. (All of the staining procedures were performed at room temperature.) Cells were first incubated with mouse anti-β-Gal (1:30) for 1 h in a humidified chamber and then with anti-mouse Cy3 (1:1000) for 45 min. The coverslips were mounted with Permount solution, air-dried, and exposed to autoradiography emulsion for at least 24 h. After development and fixation, transfected or microinjected cells were identified under a fluorescence microscope as β-Gal positive, and thymidine-positive cells among the total β-Gal-positive cells were counted. For microinjection, all β-Gal positive cells within the circle were counted, and for transfection, at least 200 β-Gal positive cells were scored for the labeling index. Cells were transfected as above although 1 μg of Topo IIα and 2 μg of AP1 or SRE luciferase constructs instead of the transfection marker were used with different cDNAs of interest or proper vectors. By the end of the experiments (48 h after DNA removal), cells were washed with phosphate-buffered saline and collected in 200 μl of reporter lysis buffer (Roche Molecular Biochemicals) with scraper. The same lysates were used for luciferase readings using a Microlite Luminometer (ML 2250, Dynateck Laboratories, Inc.) and determination of protein concentration using a Bio-Rad kit. The protein content was used to normalize luciferase activity, and similar results were also obtained with normalization to cotransfected β-galactosidase activity (data not shown). Cells were transfected and then serum-starved as described above. In some experiments, cells were treated with 50 μm PD98059 for the final 48 h, or with 20 μm SB203580 for the final 24 h. For ERK, JNK, and p38 kinase assay, cells were collected in HEPES lysis buffer (10 mm HEPES, pH 7.4, 50 mm NaF, 1% Triton X-100, 2 mm EDTA, 0.1% 2-mercaptomethanol, 2 mm Na3VO4, 2 μg/ml of aprotinin, and 1 mm phenylmethylsulfonyl fluoride) as previously published (51Waskiewicz A.J. Flynn A. Proud C.G. Cooper J.A. Mol. Cell. Biol. 1997; 16: 1909-1920Google Scholar) and used in this laboratory (38Chen G. Templeton D. Suttle D.P. Stacey D. Oncogene. 1999; 18: 7149-7160Crossref PubMed Scopus (25) Google Scholar). 0.5 ml of nuclei-free lysates were incubated with 30 μl of glutathione-agarose beads for GST-tagged ERK or -JNK, or 20 μl of anti-FLAG M2 affinity gel for FLAG-tagged p38 incubated for 4–20 h at 4 °C on a rotating plate. For immunoprecipitation of transfected HA·PRAK or Myc·MK2, cells were lysed in 25 mm HEPES, pH 7.6 buffer containing 137 mm NaCl, 3 mm EDTA, 3 mmβ-glycerophosphate, 1% Triton X-100, 0.1 mmNa3VO4, and 1 mmphenylmethylsulfonyl fluoride (21New L. Jiang Y. Zhao M. Liu K. Zhu W. Flood L.J. Kato Y. Parry G.C.N. Han J.H. EMBO J. 1998; 17: 3372-3384Crossref PubMed Scopus (272) Google Scholar). The lysates were then incubated with 2 μg of anti-HA or anti-c-Myc monoclonal antibody for 3 h and for another hour after adding 30 μl of protein A-Sepharose beads (HA·PRAK) or 20 μl of protein G-Sepharose (Myc·MK2). The precipitates were then washed two times with respective lysis buffer and two times with kinase binding buffer (20 mm HEPES, pH 7.6, 50 mm NaCl, 0.05% Triton X-100, 0.1 mm EDTA, 2.5 mmMgCl2). The kinase reaction was carried out at 30 °C for 30 min in 25 μl of kinase reaction buffer (20 mm HEPES, pH 7.6, 20 mm MgCl2, 15 μm ATP, 20 mm β-glycerophosphate, 20 mm p-nitrophenylphosphate, 0.5 mmNa3VO4, 2 mm dithiothreitol) as described previously (38Chen G. Templeton D. Suttle D.P. Stacey D. Oncogene. 1999; 18: 7149-7160Crossref PubMed Scopus (25) Google Scholar). 5 μCi of [γ-32P]ATP and 1 μg of substrate protein were used for each sample. The substrate for the kinase reaction includes MBP for ERK, GST·Jun for JNK, ATF-2 for p38, HSP27 for PRAK and MK2. Following the reaction, an equal volume of 2× Laemmli buffer was added to stop the reaction. The phosphorylated proteins were then separated in a 12.5% SDS-polyacrylamide gel. The gels were then dried, scanned, and quantitated in a phosphorimager. For each kinase assay, the transfection and the determination of the protein kinase activity were repeated two to three times with similar results. Expression of the transfected eiptope-tagged plasmids in every case was confirmed by Western blot analysis using corresponding specific antibodies. To study interactions between Ras proliferative signaling and p38, effects of oncogenic Ras on p38 activity were first examined by transient transfection assay. For this purpose, p38 kinase activity was assessed by cotransfection of FLAG-tagged wild-type p38α together with oncogenic Ras (Ha·Ras, Leu-61) or a control vector. The transfected p38 was then immunoprecipitated with an anti-FLAG (M2) antibody, and the in vitro kinase activity was determined using ATF2 as a substrate (18Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1137) Google Scholar). The ERK and JNK activities were also assayed under similar experimental conditions in which Ras was cotransfected with GST-tagged p42MAPK(ERK) or GST-tagged p54SAPK(JNK) as described previously (36Yan M. Dal T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (658) Google Scholar, 37Yan M. Templeton D.J. J. Biol. Chem. 1994; 269: 19067-19073Abstract Full Text PDF PubMed Google Scholar, 38Chen G. Templeton D. Suttle D.P. Stacey D. Oncogene. 1999; 18: 7149-7160Crossref PubMed Scopus (25) Google Scholar). As shown in Fig. 1, Ras was indeed able to activate all three MAPKs in NIH 3T3 cells, although the effect on p38 is less than with the other two kinases. These results are consistent with those reported from other laboratories (13Whitmarsh A.J. Yang S.H. Su M.S.S. Sharrocks A.D. Davis R.J. Mol. Cell. Biol. 1997; 17: 2360-2371Crossref PubMed Scopus (437) Google Scholar, 14Lin A. Minden A. Martineto H. Claret F. Lange-Carter C. Mercurio F. Johnson G.L. Karin K. Science. 1995; 268: 286-290Crossref PubMed Scopus (706) Google Scholar).Figure 3Inhibition of Ras-induced proliferation by the p38 pathway. Cells were transfected (A andB) or microinjected (C) (total DNA concentration: 150 μg/ml) with a plasmid expressing β-galactosidase together with the indicated plasmids, and the serum removed for 48 h, the final 24 h of which were in the presence of [3H]thymidine. The transfected cells were identified by immunostaining against cotransfected β-Gal, and the thymidine labeling index from at least 200 β-Gal-positive cells was then determined by autoradiography. Results shown are mean of three experiments ± S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Activation of three MAPK pathways by Ras in NIH 3T3 cells. NIH 3T3 cells were transiently transfected with oncogenic Ras (Ha·Ras, Leu-61) or control vector, together withGST-MAPKp42 (ERK), or GST-SAPKp54 (JNK), orFlag-p38α. The kinase activity following transfection was determined by an in vitro kinase assay in the GSH bead or anti-FLAG precipitates by using MBP as a substrate for ERK, GST·Jun for JNK, and ATF2 for p38. The -fold activation was calculated based on phosphorimager quantitation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To extend the observation with p38 kinase, the effect of Ras on p38 downstream kinases was then investigated. The p38-regulated protein kinase PRAK was first analyzed. To assess PRAK kinase activity, an HA epitope-tagged wild-type PRAK, a dominant-negative form of PRAK (PRAK/KM) and a mutant (PRAK/182D) in which Thr-182 was changed to aspartic acid to mimic activated PRAK (21New L. Jiang Y. Zhao M. Liu K. Zhu W. Flood L.J. Kato Y. Parry G.C.N. Han J.H. EMBO J. 1998; 17: 3372-3384Crossref PubMed Scopus (272) Google Scholar), were cotransfected with the oncogenic Ras or a control vector into NIH 3T" @default.
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• W2006569711 date "2000-12-01" @default.
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• W2006569711 title "The p38 Pathway Provides Negative Feedback for Ras Proliferative Signaling" @default.
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